Abstract
Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.
Highlights
Syphilis is a chronic sexually transmitted infection that still represents a significant burden for public health as it causes significant morbidity and mortality worldwide
The kanamycin resistance (kanR) gene sequence was derived from the Rts1 plasmid, a large conjugative plasmid originally isolated from Proteus vulgaris [18]
To ensure that non-transformed treponemes would not survive exposure to kanamycin at the concentration used for in vitro selection, the wild-type SS14 (wt-SS14) strain was propagated in media containing 25 μg/ml of kanamycin
Summary
Syphilis is a chronic sexually transmitted infection that still represents a significant burden for public health as it causes significant morbidity and mortality worldwide. The World Health Organization (WHO) estimates that syphilis global incidence ranges between 5.6 to 11 million new cases every year, while disease prevalence is between 18 to 36 million cases worldwide [1,2]. Most of those cases occur in low- and middle-income countries where the disease is endemic, syphilis rates have been steadily increasing in high-income countries for decades including in the US. Mother-to-child transmission of syphilis during pregnancy accounts for up to 50% of stillbirths in sub-Saharan Africa and a high proportion of perinatal morbidity and mortality cases [10]
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